EFFECTS OF MUTATIONS IN THE HINGE REGION OF SERPINS

An expression system for alpha1-antitrypsin in Escherichia coli was de veloped using a T7 RNA polymerase promoter. Addition of rifampicin to inhibit the E. coli RNA polymerase after induction of the T7 RNA polym erase gene resulted in about 30% of newly synthesized protein being al pha1-antitrypsin. This expression system was then used to examine the effect of mutations in the hinge region of alpha1-antitrypsin on its a ctivity. The mutations were based on ones in antithrombin III that had previously been shown to have adverse effects on activity. Mutation o f Ala347 to threonine in alpha1-antitrypsin did not affect the kinetic behavior of the protein with trypsin or human leukocyte elastase. In contrast, mutation of Gly349 to proline converted the majority of the protein into a substrate for both proteinases. The small fraction of t his mutant that was active, however, had kinetic parameters that were indistinguishable from wild-type alpha1-antitrypsin. Cleavage within t he reactive-site loop of wild-type alpha1-antitrypsin causes a conform ational change in the molecules (the S-to-R transition) and results in a marked increase in heat stability. This increase in heat stability was also seen upon cleavage within the reactive-site loops of both of the alpha1-antitrypsin mutants. The results are discussed in terms of a kinetic mechanism for serpin-proteinase interactions, in which after the formation of an initial complex the serpin partitions between the formation of a stable complex and a cleavage reaction. The hinge regi on of the reactive-site loop of serpins would appear to be important f or the partitioning in favor of the stable complex.